Every year, approximately 450,000 individuals in the United States die suddenly of cardiac arrhythmias due to disorganized ventricular conduction, with many of these deaths linked to both genetic and environmental factors. However, the identification of these genetic factors and how they work alone, together, or in concert with the environment to modulate the cellular and molecular behavior leading to arrhythmic events is far from complete. Thus, studies which identify novel genetic factors linked to cardiac arrhythmias may aid in diagnosis and treatment of patients predisposed for cardiac arrhythmias. The zebrafish has proven to be an outstanding model for understanding human diseases since it has morphologic and physiologic similarities to mammals, and provides an organism with an array of genomic tools which facilitates large-scale phenotype- based screens. For instance, there are zebrafish mutants whose phenotypes resemble complex human disorders, including common adult cardiac syndromes which result in heart failure and arrhythmias. In several cases, phenotypic similarity between man and fish has been confirmed by molecular definition. More recently, the creation of innovative transgenic tools has resulted in more sophisticated in vivo cellular and physiologic analysis as well as phenotypic-based screens. As a result, using a transgenic-based in vivo optical mapping system to perform a new physiologic-based forward genetic screen, we have assembled a collection of mutations that specifically affect ventricular conduction. Most notably, we have discovered that the dococ (dco) and daredevil (ddl) genes are critical regulators of organized ventricular conduction. dco encodes Gja3/Cx46, a gap junction protein not previously implicated in heart development or function, whereas ddl encodes RhoGa, a zebrafish ortholog of RhoG which may affect Cx46 trafficking. In contrast to cardiac Cx40, 43, and 45, the role of Cx46 in heart development or function remains to be further elucidated. Thus, we hypothesize that Cx46 functions in concert with cardiac Cx40, 43, and 45 to organize ventricular conduction through regulation of intercellular communication between specialized ventricular conduction system cardiomyocytes. Our specific aims are: 1) to elucidate underlying mechanisms of how Cx46 regulates ventricular conduction;2) to determine whether Cx46 cardiac function is conserved in the mammalian cardiac conduction system, and 3) to investigate underlying mechanisms by which rhoga regulates ventricular conduction. Overall, the combination of cellular, molecular and physiologic studies proposed in this project will provide new and in-depth insight into mechanisms of human ventricular arrhythmias. These studies may prove rewarding for prognosis and diagnosis of patients susceptible to sudden cardiac death as well as for developing therapeutic options aimed at maintaining and/or improving overall cardiac conduction. PUBLIC HEALTH RELEVANCE: Every year, approximately 450,000 individuals in the United States die suddenly of cardiac arrhythmias due to disorganized ventricular conduction, with many of these deaths linked to both genetic and environmental factors. Overall, the combination of cellular, molecular and physiologic studies proposed in this project will provide new and in-depth insight into mechanisms of human ventricular arrhythmias. These studies may prove rewarding for prognosis and diagnosis of patients susceptible to sudden cardiac death as well as for developing therapeutic options aimed at maintaining and/or improving overall cardiac conduction.